Field of Invention
This invention relates to genetically modified strains of Aureobasidium pullulans that are capable of producing desirable bioproducts. By altering the biochemistry of the disclosed strains via genetic manipulation, strains have been produced that no longer produce bio-contaminants such as melanin and melanin-related pigments. The presence of such pigments adds significant cost to the production of bioproducts because they must be removed prior to further processing of the desired products. Additionally, some strains described herein are modified to delete the genes encoding for biosynthetic enzymes that produce undesired products. Therefore, modified strains lacking the capacity of forming less-desired bioproducts have been constructed that form target bioproducts preferentially on cheaper substrates. Utilizing these methods and strains, production of desired products can be achieved under more economically feasible conditions, resulting in a benefit to food, pharmaceutical, industrial, biofuel and other important industries.
Background
Aureobasidium pullulans is a yeast-like fungus, perhaps best well-known as the source of the exopolysaccharide pullulan, which is commercially-produced for numerous consumer and industrial applications, such as emulsifiers, thickeners, and edible films (Leathers, Polysaccharides from eukaryotes. In: Vandamme, E. J., De Baets, S., and Steinbuchel, A., Editors. Biopolymers. Weinheim, Germany: Wiley-VCH. p. 1-35 (2002); Singh et al., Carb. Polymers, 73(4):515-31, (2008)). A. pullulans also produces other useful bioproducts, including industrial enzymes (Leathers et al., Biotech Lett., 35(10):1701-6 (2013); Rich et al., Enz. Microb. Tech., 53(1):33-37 (2013); Liu, et al., Anton Leeuw Int. J. G., 94(2):245-55 (2008); Leathers, J. Indus. Microbiol. 4(5):341-7 (1989); Kudanga et al., J. Basic Microbiol. 47(2): 138-47 (2007)), the biopolyester poly(β-L-malic acid) (Leathers and Manitchotpisit, Biotech. Lett., 35(1):83-9 (2013), and numerous bioactive compounds (Wang, et al., Bioresource Tech., 100(9):2639-41 (2009); Chi, et al., Appl. Microbiol. Biotech., 82(5):793-804 (2009); Slightom, et al., Gene, 431(1-2):67-79 (2009)), including an extracellular dense “oil” that accumulates on the bottom of the fermentation flask (Nagata, et al., in Biosci. Biotech. Biochem., 57(4):638-42 (1993)). A partial structure of this oil suggested that they were 3,5-dihydroxydecanoyl and 5-hydroxy-2-decanoyl esters of arabitol and mannitol (Kurosawa, et al., Biosci., Biotech. Biochem., 58(11):2057-60 (1994)) and that these polyol lipids exert an anti-proliferative effect on cancer cell lines (Isoda and Nakahara, J. Ferment. Bioeng., 84(5):403-406 (1997)).
Different strains of A. pullulans demonstrate different phenotypes with regards to bioproducts produced and can be genetically distinguished from each other. (Manitchotpisit, et al., Mycol. Res., 133(10):1107-20 (1997)). It has been noted that about half of all strains of this filamentous fungus produced oils, with certain genetically-related strains showing the highest yields (Id.; Manitchotpisit, et al., Biotech. Lett., 33(6):1151-57 (2011); Manitchotpisit, et al., World J. Micro. Biotech., 30(8):2199-2204 (2014)). Oil colors range from bright yellow to malachite and more than half of the strains produced oil that was fluorescent. Preliminary studies suggest that these pigments are likely a result of contamination by melanin, melanin breakdown products, and melanin intermediates, which are common pigments associated with A. pullulans. The oils have demonstrated biosurfactant properties (Manitchotpisit, et al. (2011)). Additionally, it has been shown that oil from different strains differentially inhibits mammalian cancer cell lines (Manitchotpisit, et al. (2014)). Recently, these microbial oils have been demonstrated to exhibit potent selective antibacterial activities against certain Streptococcal species (Bischoff, et al., J. Antibiot., (2015) 68:642-45).
The antibacterial activities of these microbial oils (also termed “liamocins”) may have potential applications, similar to other glycolipids or A. pullulans secreted metabolites, as a veterinary treatment (Cortes-Sanchez, et al., Microbiol. Res., 168(1):22-32 (2013)), an antifouling agent (Gao, et al., Marine Poll. Bull., 77(1-2):172-6 (2013); Abdel-Lateff, et al., Nat. Prod. Commns., 4(3):389-94 (2009)), and a phytopathogen control agent (Le Dang, et al., J. Agr. Food Chem., 62(15):3363-70 (2014)). In addition, the medium-chain dihydroxydecanoate fatty acid is promising as a potential chemical feedstock for the synthesis of a wide variety of commercially relevant products, such as biosurfactants and polymers (Tang, et al., Polymer Chem., 5(9):3231-37 (2014); Schneiderman, et al., J. Chem. Edu., 91(1); 131-5 (2014).
A. pullulans is also used for production the antibiotic aureobasidin and β-glucan. Production of both of these products could be improved by utilizing strains that are no longer producing melanin or melanin-related pigments. In addition, A. pullulans was recently shown to produce significant amounts of intracellular lipids (Wang et al., Process Biochem., 49(5):725-31 (2014)), which may have potential for biodiesel or specialty oil production (Sitepu et al., Biotech. Adv., 32(7):1336-1360 (2014)).
As alluded to above, one of the problems with using A. pullulans for bioproduct formation is that most strains produce dark melanin-associated pigments that contaminate the final desired product. These contaminating pigments can be removed by treatment with activated charcoal or hydrogen peroxide, but further purification with ultrafiltration and ion exchange resins are typically required. These clean up steps frequently result in loss of the desired bioproduct and add further cost to the manufacturing process (Mishra and Vuppu, Res. J. Microbiol. Biotech., 2:16-19 (2013); Leathers, Appl. Microbiol. Biotech., 62(5-6):468-473 (2003)). Oils and other bioproducts free of melanin or melanin-related pigments would be easier and cheaper to purify, making them more valuable and more economically feasible to produce. Thus, downstream products, such as biodiesel and food additives and preservatives would also be cheaper to produce.
Therefore, it is an object of the present invention to provide methods and strains of A. pullulans for the production of desired bioproducts as well as melanin-free bioproducts. Using the strains and methodologies presented here provides not only for a cheaper alternative to the use of non-modified strains, but also allows for a more ecologically responsible approach for the production of some bioproducts.